Methods for inhibiting an immune response by blocking the GP39/CD40 and CTLA4/CD28/B7 pathways and compositions for use therewith

ABSTRACT

The present invention provides a method for inhibiting an immune reponse and a method for inhibiting rejection of transplanted tissues. This method comprises preventing an endogenous molecule on a cell selected from the group consisting of gp39 and CD40 antigens from binding its endogenous ligand and preventing an endogenous molecule on a cell selected from the group consisting of CTLA4, CD28, and B7 antigens from binding its endogenous ligand. The prevention of such molecules from binding their ligand thereby blocks two independent signal pathways and inhibits the immune response resulting in transplanted tissue rejection.

[0001] This application is based on United States provisional patentapplication Ser. No. 60/013,751 filed on Mar. 20, 1996.

[0002] Throughout this application various publications are referenced.The disclosures of these publications in their entireties are herebyincorporated by reference into this application in order to more fullydescribe the state of the art to which this invention pertains.

BACKGROUND OF THE INVENTION

[0003] CD28 is expressed on most T lineage cells and plasma cells (June,C. H. et al., Immunol. Today 11, 211-16 (1990); Damle et al., Proc.Natl. Acad. Sci. 78:5096-6001 (1981)). The ligand for CD28 is B7, whichis expressed on activated B cells (Linsley, P. S. et al., Proc. Natl.Acad. Sci. USA 87, 5031-35 (1990); Linsley, P. S. et al., J. Exp. Med.173, 721-730 (1991).

[0004] CD40 is a member of the tumor necrosis factor receptor (TNFR)family of type I membrane-bound signaling receptors. Though originallyidentified as a B cell antigen, CD40 is expressed by all antigenpresenting cells (APC) including dendritic cells, monocytes, and Bcells.

[0005] The ligand for CD40 is gp39, which binds to CD40 and thus canactivate B cells. Gp39 is also known as CD40L, TRAP and T-BAM. Gp39 is atype II cell surface protein with significant homology to TNF and istransiently expressed by activated T cells. In addition to T cells, gp39is expressed by basophils, mast cells, and eosinophils.

[0006] The CD28 and CD40 pathways play essential roles in the initiationand amplification of T-dependent immune responses (Bluestone, J. A.Immunity 2, 555-9 (1995); Banchereau J., et al. Own. Rev. Immunol. 12,881-922 (1994); Durie, F. H., et al. Science 261, 1328-30 (1993); Foy,T. M., et al. J Exp Med 178, 1567-75 (1993); Van den Eertwegh, A. J. M.,et al. J Exp Med 178, 1555-65 (1993)).

[0007] CD28/B7 interactions provide critical “second signals” necessaryfor optimal T cell activation, and IL-2 production (Jenkins, M. K., etal. J. Immunol. 147, 2461-6 (1991); Schwartz, R. H. Cell 71, 1065-8(1992); Boussiotis, V. A., et al. J. Exp. Med. 178, 1753-1763 (1993)),whereas CD40/gp39 signals provide costimulation for B cell, macrophage,endothelial cell, and T cell activation (Grewal, I. S., et al. Nature378, 617-620 (1995); van Essen, D., et al. Nature 378, 620-623 (1995);Hollenbaugh, D., et al. J. Exp. Med. 182, 33-40 (1995); Armitage, R. J.,et al. Nature 357, 80-2 (1992); Cayabyab, M., et al. J. Immunol. 152,1523-31 (1994); Noelle, R., et al. Proc. Natl. Acad. Sci. USA 89,6550-6554 (1992); Alderson, M. et al. J. Exp. Med. 178, 669-674 (1993)).

[0008] Most immune responses often cause rejection of transplantedtissues and organs. Thus, inhibition of those immune responses arecritical in the success of tissue transplantation. There have beenstudies aimed at blocking either of the CD28 or CD40 pathways, however,blockade of either of these pathways alone has not been sufficient topermit engraftment of highly immunogenic allografts (Turka, L. A., etal. Proc. Nat'l Acad. Sci. USA 89, 11102-11105 (1992); Parker, D. C., etal. Proc. Nat'l Acad. Sci. USA 92, 9560-9564 (1995); Larsen, C. P. etal. Transplantation 61, 4-9 (1996)). The monotherapies blocking eitherCD28 or CD40 pathway only resulted in at best temporary, and sometimeslonger, periods of survival of transplanted tissues. Neither blockadealone uniformly promoted graft survival.

[0009] The vigorous immune response to xenogeneic organ transplants hasserved as a powerful barrier to the application of this technique toclinical transplantation (Platt J. L., Curr. Opin. Imm. 8, 721-728(1996). Previous experimental attempts to prolong xenogeneic skin graftshave required either whole body irradiation followed by mixedxeno/syngeneic reconstitution (Ildstad S. T., Sachs D. H., Nature, 307:168-170 (1984)), or rigorous preconditioning with thymectomy combinedwith depleting anti-T cell antibodies (Pierson III R. N., Winn H. J.,Russell P. S., Auchincloss Jr. H., J. Exp. Med., 170:991-996 (1989); andSharabi Y, Aksentijevich I., Sundt III T. M., Sachs D. H., Sykes M., J.Exp. Med., 172:195-202 (1990). These Strategies have recently been usedto promote skin graft acceptance across a discordant xenogeneic barrier(Zhao Y., Swenson K., Sergio J., Arn J. S., Sachs D. H., Sykes M., Nat.Med., 2(11):1211-1216 (1996)). However, the potential morbidityassociated with cytoablative treatment regimens present a significantobstacle to the introduction of these strategies into use in clinicalsolid organ transplantation. Thus the development of non-cytoablativestrategies to prolong xenograft survival would greatly facilitate theclinical application of these techniques.

[0010] Presently, there exists a need to provide ways to effectlong-term tolerance of transplanted tissues by the host, therebyincreasing the survival rate of transplantation. To do so, it isnecessary to ensure sufficient immunologic unresponsiveness in thetransplant recipient.

[0011] We have found that the inhibition of T-dependent immune responsesresulting from blockade of either CD28 or CD40 signals is potent, butincomplete. The data herein demonstrate that simultaneous blockade ofthese pathways unexpectedly inhibits acute and chronic rejection oftransplanted tissue in vivo. Independent blockade of these pathwaysusing a soluble CTLA4 molecule or antibodies which recognize and bindgp39 failed to even minimally prolong survival of primary skintransplanted tissue.

[0012] The invention herein involves the discovery that simultaneousblockade of CD28 and CD40 signals promoted long-term survival of fullyallogeneic as well as xenogeneic skin grafts. Prolongation of skinallograft survival was eliminated by cyclosporine A (CyA), suggestingthat it is an active process requiring intact signaling via the TcR/CD3complex and/or other CyA sensitive pathways. Moreover, CTLA4Ig/MR1promoted long-term acceptance of primarily vascularized cardiac grafttissue, and inhibited the development of chronic vascular rejection.

[0013] The effect demonstrated in the two transplantation models hereinindicates that CD28 and CD40 provide interrelated, yet independentsignaling pathways required for the generation of effective T cellresponses. This discovery provides methods which are new and moreeffective strategies to manipulate immune responses includingsuppressing graft rejection.

SUMMARY OF THE INVENTION

[0014] The present invention provides a method for inhibiting rejectionof a transplanted tissue. This method comprises preventing an endogenousmolecule (e.g., antigen) on a cell selected from the group consisting ofgp39 and CD40 from binding its endogenous ligand and preventing anendogenous molecule on a cell selected from the group consisting ofCTLA4, CD28, and B7 from binding its endogenous ligand. The preventionof such molecules from binding their ligands thereby blocks twoindependent signal pathways and inhibits the immune response responsiblefor transplanted tissue rejection.

[0015] Further, the invention provides a method for inhibiting an immuneresponse involved with transplanted tissue rejection comprisingcontacting a B7-positive cell with a first soluble ligand whichrecognizes and binds the B7 antigen, and contacting a gp39-positive cellwith a second soluble ligand which recognizes and binds the gp39antigen. The binding of the B7-positive cell to the first soluble ligandblocks the reaction of the B7 antigen with endogenous CTLA4 or CD28.Additionally, the binding of the gp39 antigen to the second solubleligand blocks the reaction of gp39 antigen with endogenous CD40. Thisblockage of both the gp39 and B7 pathways inhibits immune responses.

[0016] Applicants' discovery includes a method for inhibiting immuneresponses mediated by the gp39 and B7 pathways in a subject. This methodcomprises administering to the subject a first soluble ligand whichrecognizes and binds the B7 antigen and a second soluble ligand whichrecognizes and binds the gp39 antigen.

[0017] The binding of both the first and second soluble ligands to theirreceptors inhibits the immune response mediated by the gp39 and B7pathways by preventing an endogenous molecule on a cell selected fromthe group consisting of gp39 and CD40 antigens from binding itsendogenous ligand and preventing an endogenous molecule on a cellselected from the group consisting of CTLA4, CD28, or B7 from bindingits ligand.

[0018] The present invention also provides a method for inhibitingtransplant rejection in a subject. This method comprises administeringto the subject an effective amount of a combination of a first solubleligand which recognizes and binds the B7 antigen on B7-positive cellsand a second soluble ligand which recognizes and binds the gp39 antigenon gp39-positive cells.

[0019] The binding of B7-positive cells with the first soluble ligandand gp39-positive cells with the second soluble ligand disruptsendogenous CTLA4-, CD28-, and gp39-positive cell interactions withB7-positive cells and gp39-positive cells so that transplant rejectionis inhibited.

BRIEF DESCRIPTION OF THE FIGURES

[0020]FIG. 1 is a bar graph showing that simultaneous blockade of CD28and CD40 signals ablate popliteal lymph node alloimmune responses invivo.

[0021]FIG. 2A is a line graph that shows CTLA4Ig/MR1 treatment prolongscardiac allograft survival in comparison with CTLA4Ig or MR1 alone.

[0022]FIG. 2B is a photograph of a histologic section showingCTLA4Ig-treated cardiac allograft at day 62 having extensive lymphocyticinfiltration, interstitial fibrosis, and severe coronary arterialintimal thickening and fibrosis consistent with chronic rejection (leftpanel 100×magnification; right panel 400×magnification).

[0023]FIG. 2C is a photograph of a histologic section showing aMR1-treated cardiac allograft at day 62 having less lymphocyticinfiltration and interstitial fibrosis, but severe coronary vasculopathycharacteristic of chronic rejection (left panel 100×magnification; rightpanel 400× magnification).

[0024]FIG. 2D is a photograph of a histologic section ShowingCTLA4Ig/MR1-treated cardiac allografts at day 58, free from lymphocyticinfiltration, fibrosis, coronary arterial intimal lesions (left panel100×magnification; right panel 400×magnification).

[0025]FIG. 2E is a photograph of a histologic section showing normaluntransplanted BALB/c hearts (left panel 100×magnification; right panel400×magnification).

[0026]FIG. 3A is a photograph of ethidium bromide stained gel stripsshowing intragraft expression of immune mediator transcripts usingRT-PCR in untreated, MR1 treated, CTLA4Ig treated, and MR1/CTLA4Igtreated cardiac allografts.

[0027]FIG. 3B is a series of bar graphs showing the mean PCR productband intensities±standard deviation.

[0028]FIG. 4A is a line graph showing data of mice treated with MR1alone, CTLA4Ig alone, and a combination of MR1 and CTLA4Ig.

[0029]FIG. 4B is a line graph showing data of mice treated with CyA, CyAand CTLA4Ig, and CyA and MR1.

[0030]FIG. 4C is a line graph showing t he effects of perioperativetreatment with YTS191 and MR1, alone, MR1 and CTLA4Ig, and YTS191 andCTLA4Ig on primary skin allografts.

[0031]FIG. 4D is a photograph showing healthy appearance of a BALB/cskin graft on a CTLA4Ig/MR1 treated recipient.

[0032]FIG. 4E is a photograph showing a control allograft undergoingrejection.

[0033]FIG. 4F is a photograph of a histologic section of a skin graftshowing healthy appearance of an accepted graft at 100 days aftertransplant showing well preserved epidermis hair follicles and adnexalstructures.

[0034]FIG. 4G is a photograph showing a BALB/c skin graft on anuntreated recipient eight days after transplant. The graft showsextensive lymphocytic infiltrate.

[0035]FIG. 5A is a series of line graphs showing the effects in vitro ofusing MR1 alone, CTLA4Ig alone, and a combination of MR1/CTLA4Ig onthree different cell populations.

[0036]FIG. 5B is a series of bar graphs showing the effects in vivo ofusing MR1 alone, CTLA4Ig alone, and a combination of MR1/CTLA4Ig.

[0037]FIG. 6A is a bar graph showing weight of the immunized popliteallymph node relative to the contralateral node in C3H mice in response tofoot pad immunization with irradiated (2000 RADS) rat (Sprague-Dawley)splenocytes. Human IgG (stippled), CTLA4-Ig (gray), MR1 (white),CTLA4-Ig/MR1 (black), normal unimmunized node (hatched).

[0038]FIG. 6B is a line graph showing in vitro proliferation of lymphnode cells after harvesting the popliteal lymph at five days afterimmunization. Human IgG (stippled), CTLA4Ig (gray), MR1 (white),CTLA4-Ig/MR1 (black), normal unimmunized node (hatched).

[0039]FIG. 6C is a bar graph showing that simultaneous blockade of theCD40 and CD28 pathways markedly inhibits cytokine production of IL-2.Human IgG (stippled), CTLA4-Ig (gray), MR1 (white), CTLA4-Ig/MR1(black), normal unimmunized node (hatched).

[0040]FIG. 6D is a bar graph showing that simultaneous blockade of theCD40 and CD28 pathways markedly inhibits cytokine production of INFγ.Human IgG (stippled), CTLA4-Ig (gray), MR1 (white), CTLA4-Ig/MR1(black), normal unimmunized node (hatched).

[0041]FIG. 7A is a line graph showing that C3H recipients treated withCTLA4-Ig (500 μg) on days 0, 2, 4 and 6 combined with MR1 (500 μg) ondays 0, 2, 4 and 6 had prolonged survival of Sprague-Dawley rat cardiacallografts.

[0042]FIG. 7B is a photograph of an untreated cardiac xenograft at day 6showing widespread tissue destruction (400×).

[0043]FIG. 7C is a photograph of a CTLA4-Ig treated cardiac xenograft atday 20 showing lymphocytic infiltration, myocyte destruction, andcoronary vasculopathy (400×).

[0044]FIG. 7D is a photograph of a MR1 treated cardiac xenograft at day20 showing lymphocytic infiltration, myocyte destruction, and coronaryvasculopathy (400×).

[0045]FIG. 7E is a photograph of a normal untransplanted Sprague-Dawleyrat heart (400×).

[0046]FIG. 7F is a photograph of a CTLA4-Ig/MR1 treated cardiacxenograft at day 20, essentially free from lymphocytic infiltration andfibrosis (400×).

[0047]FIG. 7G is a photograph of a CTLA4-Ig/MR1 treated cardiacxenograft at day 122, demonstrating excellent preservation of bothmyocytes and vascular structures (400×).

[0048]FIG. 8A is a series of line graphs showing prolongation ofSprague-Dawley rat skin xenograft survival in C3H mice treated with MR1and CTLA4-Ig administered together in the perioperative period ascompared with xenograft recipients treated with either MR1 alone orCTLA4-Ig alone and untreated controls.

[0049]FIG. 8B is a series of line graphs showing no significant changein xenograft survival following chronic treatment (beginning after thestandard 4 dose regimen) with either the CTLA4-Ig/MR1 combination orMR1.

[0050]FIG. 8C is a photograph showing the healthy appearance of aSprague-Dawley rat skin graft on a CTLA4-Ig/MR1 treated C3H recipient at100 days after transplant.

[0051]FIG. 8D is a photograph showing a control xenograft of skinundergoing rejection at 10 days post transplant.

[0052]FIG. 8E is a photograph of a hematoxylin-eosin stained histologicsection of an accepted CTLA4-Ig/MR1 treated graft at 50 days aftertransplant showing well-preserved histologic architecture (400×).

[0053]FIG. 8F is a photograph of a hematoxylin-eosin stained histologicsection of a Sprague-Dawley rat skin graft on an untreated C3H recipient8 days after transplant showing extensive lymphocytic infiltrates(400×).

[0054]FIG. 9A is a scatter plot showing ablation of evoked xenoantibodyresponse in serum collected from C3H recipients 55 days after skinxenografts from Sprague-Dawley donors. Control mice that had received notreatment had readily detectable IgG xenoantibody. Either CTLA4-Ig orMR1 alone partially blocked the xenoantibody response. The combinationof CTLA4-Ig and MR1 essentially ablated the evoked xenoantibodyresponse. Each data point represents the analysis of an individualrecipient.

[0055]FIG. 9B is a scatter plot showing ablation of evoked xenoantibodyresponse in serum collected from C3H recipients days after heartxenografts from Sprague-Dawley donors.

DETAILED DESCRIPTION OF THE INVENTION

[0056] Definitions

[0057] All scientific and technical terms used in this application havemeanings commonly used in the art unless otherwise specified. As used inthis application, the following words or phrases have the meaningsspecified.

[0058] As used herein “monoclonal antibodies directed against gp39 ” or“anti-gp39” includes MR1. Anti-gp39 is also known in the literature asan antiCD40 ligand. Examples of MR1 include, but are not limited tomonoclonal antibodies directed against gp39 from mouse; antibodiesdirected against gp39 from other species such as monkey, sheep, humanare included. Additionally, “monoclonal antibodies directed againstgp39” or “anti-gp39” includes any antibody molecule, fragment thereof,or recombinant binding protein that recognizes and binds gp39.

[0059] As used herein, “administering” means oral administration,administration as a suppository, topical contact, intravenous,intraperitoneal, intramuscular or subcutaneous administration, or theimplantation of a slow-release device such as a miniosmotic pump, to thesubject.

[0060] As used herein, “pharmaceutically acceptable carrier” includesany material which when combined with the antibody retains theantibody's immunogenicity and is non-reactive with the subject's immunesystems. Examples include, but are not limited to, any of the standardpharmaceutical carriers such as a phosphate buffered saline solution,water, emulsions such as oil/water emulsion, and various types ofwetting agents. Other carriers may also include sterile solutions,tablets including coated tablets and capsules.

[0061] Typically such carriers contain excipients such as starch, milk,sugar, certain types of clay, gelatin, stearic acid or salts thereof,magnesium or calcium stearate, talc, vegetable fats or oils, gums,glycols, or other known excipients. Such carriers may also includeflavor and color additives or other ingredients. Compositions comprisingsuch carriers are formulated by well known conventional methods.

[0062] As used herein, “transplanted tissue” includes autografts,isografts, allografts, and xenografts. Examples of transplanted tissueinclude, but are not limited to, solid organ transplants such as heart,liver or kidney, skin grafts, pancreatic islet cells, bone marrow graftsor cell suspensions.

[0063] As used herein, “B7 ” includes, B7-1 (also called CD80), B7-2(also called CD86), B7-3, and the B7 family, e.g., a combination ofB7-1, B7-2 and/or B7-3.

[0064] In order that the invention herein described may be more fullyunderstood, the following description is set forth.

[0065] The discovery herein is related to a method for inhibitingrejection of a transplanted tissue. In one embodiment, the methodcomprises preventing an endogenous molecule on a cell selected from thegroup consisting of gp39 and CD40 from binding its endogenous ligand.The method provides preventing an endogenous molecule on a cell selectedfrom the group consisting of CTLA4, CD28, and B7 from binding itsendogenous ligand. The prevention of these molecules from binding theirendogenous ligands blocks two independent signal pathways. The blockageof these two independent signal pathways inhibits the immune responsesthat cause transplanted tissue rejection.

[0066] In one example of the invention, endogenous gp39 antigen isprevented from binding its endogenous ligand. This example comprises thestep of contacting a gp39-positive cell with a soluble ligand whichrecognizes and binds the gp39 antigen (e.g., by using soluble ligandssuch as MR1 or other antibodies which bind gp39, and soluble CD40molecules).

[0067] This example comprises the additional step of preventing theendogenous CTLA4 antigen from binding its endogenous ligand. Thiscomprises the step of contacting a B7-positive cell with a solubleligand which recognizes and binds the B7 antigen such as CTLA4Ig (U.S.Pat. No. 5,434,131, issued Jul. 18, 1995), the BB-1 monoclonal antibodyor other antibodies directed against B7.

[0068] The binding of the gp39-positive cell to its soluble ligandblocks the reaction of endogenous gp39 antigen with endogenous CD40. Thebinding of the B7-positive cell to its soluble ligand blocks thereaction of the endogenous B7 antigen with endogenous CTLA4 and CD28.This combined blockage inhibits the immune response.

[0069] In another example, endogenous CD40 antigen is prevented frombinding its endogenous ligand. This example comprises the step ofcontacting a CD40-positive cell with a soluble ligand which recognizesand binds the CD40 antigen. Suitable ligands include antibodies directedagainst CD40 or soluble gp39 (sgp39).

[0070] This example comprises the additional step of preventing theendogenous CTLA4 antigen from binding its endogenous ligand. This stepcomprises contacting a B7-positive cell with a soluble ligand whichrecognizes and binds the B7 antigen examples of this soluble ligandinclude CTLA4Ig, soluble CD28 molecules, and antibodies directed againstB7.

[0071] The binding of the CD40-positive cell to its soluble ligandblocks the reaction of endogenous CD40 antigen with endogenous gp39. Thebinding of the B7-positive cell to its soluble ligand blocks thereaction of the B7 antigen with endogenous CTLA4. The combined blockageinhibits the immune response.

[0072] In yet another example, endogenous gp39 antigen is prevented frombinding its endogenous ligand as described above. The example comprisesthe additional step of preventing the endogenous CD28 antigen frombinding its endogenous ligand. This step comprises contacting aB7-positive cell with a soluble ligand which recognizes and binds the B7antigen. Examples include CTLA4Ig, soluble CD28 molecules, andantibodies directed against B7 such as BB-1.

[0073] The binding of the gp39-positive cell to its soluble ligandblocks the reaction of gp39 antigen with endogenous CD40. The binding ofthe B7-positive cell to its soluble ligand blocks the reaction of the B7antigen with endogenous CD28. This combined blockage inhibits the immuneresponse.

[0074] In another example, endogenous CD40 antigen is prevented frombinding its endogenous ligand as described above. The example providesthe additional step of preventing the endogenous B7 antigen from bindingits endogenous ligand. This comprises contacting a CD28-positive cellwith a soluble ligand which recognizes and binds the CD28 antigen.Examples of such soluble ligands include soluble B7 molecules andantibodies directed against CD28.

[0075] The binding of the CD40-positive cell to the soluble ligandblocks the reaction of CD40 antigen with endogenous gp39. The binding ofthe CD28-positive cell to the soluble ligand blocks the reaction of theB7 antigen with endogenous CD28. This combined blockage inhibits theimmune response.

[0076] In yet another example, endogenous CD40 antigen is prevented frombinding its ligand as described above. This example provides theadditional step of preventing the endogenous B7 antigen from binding itsendogenous ligand which comprises contacting a CTLA4-positive cell witha soluble ligand which recognizes and binds the CTLA4 antigen. Examplesof such soluble ligands include soluble B7 molecules and antibodiesdirected against CTLA4.

[0077] The binding of the CD40-positive cell to the soluble ligandblocks the reaction of CD40 antigen with endogenous gp39. Additionally,the binding of the CTLA4- or CD28-positive cell to the soluble ligandblocks the reaction of the CTLA4 antigen with endogenous B7. Thiscombined blockage inhibits the immune response.

[0078] In a further example, endogenous CD40 antigen is prevented frombinding its ligand as described above. This example provides theadditional step of preventing the endogenous B7 antigen from binding itsendogenous ligand which comprises contacting a CD28-positive cell with asoluble ligand which recognizes and binds the CD28 antigen. Examples ofsuch soluble ligands include soluble B7 molecules and antibodiesdirected against CD28.

[0079] The binding of the CD40-positive cell to the soluble ligandblocks the reaction of gp39 antigen with endogenous CD40. Further, thebinding of the CD28-positive cell to the soluble ligand blocks thereaction of the CD28 antigen with endogenous B7. This combined blockageinhibits the immune response.

[0080] Also, in another example, endogenous CD40 antigen is preventedfrom binding its ligand as described above. This example provides theadditional step of preventing the endogenous B7 antigen from binding itsendogenous ligand which comprises contacting a CTLA4-positive cell witha soluble ligand which recognizes and binds the CTLA4 antigen.

[0081] The binding of the CD40-positive cell to the soluble ligandblocks the reaction of gp39 antigen with endogenous CD40. Additionally,the binding of the CTLA4-positive cell to the soluble ligand blocks thereaction of the CTLA4 antigen with endogenous B7. This combined blockageinhibits the immune response.

[0082] Additionally, the present invention provides another embodimentfor a method for inhibiting an immune response resulting in graftrejection. This embodiment comprises contacting a B7-positive cell witha first soluble ligand which recognizes and binds the B7 antigen, andcontacting a gp39-positive cell with a second soluble ligand whichrecognizes and binds the gp39 antigen.

[0083] The binding of the B7-positive cell to the first soluble ligandblocks the reaction of the B7 antigen with endogenous CTLA4 or CD28.Further, the binding of the gp39 antigen to the second soluble ligandblocks the reaction of gp39 antigen with endogenous CD40. Thecombination of this blockage inhibits the immune response.

[0084] Additionally, the invention provides a method for inhibiting animmune response mediated by the CTLA4/CD28/B7 and gp39/CD40 pathways ina subject. In accordance with the practice of the invention, the subjectmay be an animal subject such as a human, a dog, a cat, a sheep, ahorse, a mouse, a pig, or a cow.

[0085] The method comprises administering to the subject a first solubleligand which recognizes and binds the B7 antigen (e.g. soluble CTLA4 orCD28 molecules) and a second soluble ligand which recognizes and bindsthe gp39 antigen (e.g., monoclonal antibodies directed against gp39(MR1) or soluble CD40 molecules). The binding of the first and secondligands to their receptor inhibits the immune response mediated byCTLA4-, CD28-, and gp39- cell interactions with B7- and CD40-positivecells.

[0086] Also, the invention provides a method for inhibiting transplantrejection in a subject. This method comprises administering to thesubject an effective amount of a combination of a first soluble ligandwhich recognizes and binds the B7 antigen on B7-positive cells and asecond soluble ligand which recognizes and binds the gp39 antigen ongp39-positive cells. The binding of B7-positive cells with the firstsoluble ligand and gp39-positive cells with the second soluble liganddisrupts endogenous CTLA4-, CD28-, and gp39- cell interactions withB7-positive cells and gp39-positive cells so that transplant rejectionis inhibited.

[0087] In accordance with the practice of the invention, the firstsoluble ligand may be a recombinant binding molecule having at least aportion of the extracellular domain of CTLA4. In accordance with thepractice of the invention, the extracellular portion of CTLA4 is joinedto a non-CTLA4 protein sequence. The non-CTLA4 protein sequence may beat least a portion of an immunoglobulin molecule.

[0088] In one specific example of the invention, the ligand is CTLA4Igfusion protein, e.g., the CTLA4Ig fusion protein deposited with theAmerican Type Culture Collection (ATCC) in Rockville, Md., under theprovisions of the Budapest Treaty on May 31, 1991 and accorded ATCCaccession number: 68629. Alternatively, the ligand may be aCD28Ig/CTLA4Ig fusion protein hybrid (U.S. Pat. No. 5,434,131, issuedJul. 18, 1995).

[0089] In an alternative embodiment, the first soluble ligand may be amonoclonal antibody reactive with B7 antigen, e.g., the antibody may beanti-BB1 monoclonal antibody (Clark et al., Human Immunol. 16:100-113(1986); Yokochi et al., J. Immunol. 128:823 (1981)); Freeman et al. (J.Immunol. 143(8):2714-2722 (1989); and Freedman et al., J. Immunol.139:3260 (1987)).

[0090] In another embodiment, the ligand may be a CD28Ig/CTLA4Ig fusionprotein hybrid having a first amino acid sequence corresponding to aportion of the extracellular domain of CD28 receptor fused to a secondamino acid sequence corresponding to a portion of the extracellulardomain of CTLA4 receptor and a third amino acid sequence correspondingto the hinge, CH2 and CH3 regions of human immunoglobulin Cγ1.

[0091] In one embodiment of the invention, the second soluble ligand forthe gp39 antigen may be a monoclonal antibody reactive with the gp39antigen, e.g., the MR1 anti-murine monoclonal antibody or the anti-humangp39 antibody (U.S. Pat. No. 5,474,771, issued Dec. 12, 1995).

[0092] In another embodiment of the invention, the method comprisesadministering to the subject a soluble fusion protein, the solublefusion protein comprising a first binding domain and a second bindingdomain.

[0093] In one example, the first binding domain is a ligand whichrecognizes and-binds the gp39 antigen. Examples include CD40 andmonoclonal antibodies directed against gp39. In another example, thefirst binding domain is a ligand which recognizes and binds the CD40antigen. Examples include gp39 and monoclonal antibodies directedagainst CD40.

[0094] In one example, the second binding domain is a ligand whichrecognizes and binds CTLA4. Examples include B7 and monoclonalantibodies directed against CTLA4. In another example, the secondbinding domain is a ligand which recognizes and binds the CD28 antigen.Examples include B7 and monoclonal antibodies directed against CD28. Inanother example, the second binding domain is a ligand which recognizesand binds the B7 antigen. Examples include CTLA4, CD28 and monoclonalantibodies directed against B7.

[0095] Soluble ligands may be administered during transplant, beforetransplant, or after transplant. Soluble ligands may be administered byoral means, transdermal means, intravenous means, intramuscular means,intraperitoneal, or by subcutaneous administration.

[0096] The most effective mode of administration and dosage regimen forthe molecules of the present invention depends upon the location of thetissue or disease being treated, the severity and course of the medicaldisorder, the subject's health and response to treatment and thejudgment of the treating physician. Accordingly, the dosages of themolecules should be titrated to the individual subject.

[0097] By way of example, the interrelationship of dosages for animalsof various sizes and species and humans based on mg/m² of surface areais described by Freireich, E. J., et al. Cancer Chemother., Rep. 50 (4):219-244 (1966). Adjustments in the dosage regimen may be made tooptimize suppression of the immune response resulting in graftrejection, e.g., doses may be divided and administered on a daily basisor the dose reduced proportionally depending upon the situation (e.g.,several divided doses may be administered daily or proportionallyreduced depending on the specific therapeutic situation).

[0098] It would be clear that the dose of the composition of theinvention required to achieve an appropriate clinical outcome may befurther reduced with schedule optimization.

[0099] The present invention also provides pharmaceutical compositionsuseful in inhibiting graft rejection or in inhibiting an immuneresponse. In one embodiment, these compositions comprise an effectiveamount of a combination of (a) soluble ligands which recognize and bindany one of CTLA4, CD28, and B7 antigens, together with (b) solubleligands which recognize and bind any one of gp39 and CD40 antigens andan acceptable carrier. In another embodiment, these compositionscomprise an effective amount of a soluble fusion protein comprising afirst binding domain and a second binding domain, wherein the firstbinding domain is a ligand which recognizes and binds any one of gp39 orCD40 antigens and the second binding domain is a ligand which recognizesand binds any one of CTLA4, CD28, and B7 antigens.

[0100] ADVANTAGES OF THE INVENTION: Despite the many advances inclinical immunosuppression, chronic vascular rejection remains the majorsource of transplant failure for which there remains no effectivetherapy. The experiments described herein show that blocking theCD28/CTLA4/B7 and gp39/CD40 pathways inhibits the development of chronictransplant vasculopathy in transplanted tissues. These data show thatimmune responses to allogeneic and xenogeneic grafts can be inhibitedwithout cytoablation. When compared to the use of soluble CTLA4molecules alone, the use of soluble CTLA4 molecules together with asoluble ligand that recognizes and binds gp39 provides dramaticallyprolonged immunosuppression.

[0101] the following examples are presented to illustrate the presentinvention and to assist one of ordinary skill in making and using thesame. The examples are not intended in any way to otherwise limit thescope of the invention.

EXAMPLE 1

[0102] The data in this example show that simultaneous blockade of CD28and CD40 signals ablates popliteal lymph node alloimmune responses invivo.

[0103] Method

[0104] Male C3H/HeJ (The Jackson Laboratory, Bar Harbor, Me.) mice weresubcutaneously immunized with 2×10⁶ BALB/c splenocytes in 50 μl ofsterile normal saline in the left foot pad and 50 μl of sterile normalsaline in the right foot pad on day 0 and then treated intraperitoneallywith MR1 (250 μg), CTLA4Ig (250 μg), or both reagents on days 0, 2 and4.

[0105] The mice were sacrificed on day 5, the popliteal lymph nodes wereharvested using an operating microscope (20×magnification) and the freshweight of each node was determined to the nearest 0.1 mg with ananalytical balance (Model A-160, Denver Instrument Company, Arvada,Colo.).

[0106] Discussion

[0107] Five days after subcutaneous immunization with allogeneicsplenocytes, the draining popliteal lymph nodes on the side of antigenchallenge underwent a >5 fold increase in weight relative to thecontralateral node in untreated control mice. Treatment with eitherCTLA4Ig or MR1 resulted in a 50-60% inhibition of the response, whereasconcomitant administration of CTLA4Ig and MR1 ablated lymph nodeexpansion in response to antigen challenge. The results represent themean±standard deviation for 3 individual mice in each group. Similarresults were obtained in three independent experiments.

[0108] Control mice demonstrated a 4-6 fold increase in the weight ofthe node draining the immunized foot relative to the node draining thecontralateral foot injected with sterile saline (FIG. 1). This increasein weight was accompanied by a dramatic expansion of the lymphocyte-richparacortical (T cell) and cortical (B cell) regions. When administeredalone, CTLA4Ig and MR1 each produced partial inhibition of this response(57% and 56% inhibition, respectively). The combination of CTLA4Ig/MR1ablated lymph node expansion (98% inhibition, FIG. 1) and preventedexpansion of the paracortical and lymphoid follicles.

EXAMPLE 2

[0109] This example shows prolongation of cardiac allograft survival andinhibition of vasculopathy associated with chronic rejection.

[0110] Method

[0111] Male C3H/HeJ mice were transplanted with primarily vascularizedBALB/c heart allografts at 8-12 weeks of age using microsurgicaltechniques (Corry, R. J., Winn, H. J. & Russell, P. S. Transplantation16, 343-350 (1973)).

[0112] Rejection was defined by the loss of palpable cardiaccontractions with confirmation at laparotomy by direct visualization. Atspecified times after transplant, the transplanted hearts were excised,formalin fixed and embedded in paraffin. Tissue sections (5 μm) werestained with Masson's Trichrome or hematoxylin-eosin. Each histologicspecimen was reviewed by a cardiac transplant pathologist (KJW) blindedto the treatment modality.

[0113] Discussion

[0114] In FIG. 2A C3H/HeJ recipients were treated with CTLA4Ig (200μg/dose) on days 0, 2, 4 and 6 combined with MR1 (250 μg/dose) on days0, 2 and 4, and had long term survival of BALB/c cardiac allografts(Median Survival Time (MST) >70 days, n=7). The control groups includedrecipients treated with: CTLA4Ig alone (MST=50 days, n=12); MR1 aloneMST=70 days, n=12); and no treatment (MST=12 days, n=7).

[0115] All recipients were followed for 70 days with the exception ofthree mice with surviving transplants in each experimental group whichwere sacrificed for histologic analysis at 58-63 days post transplant.

[0116] In FIG. 2B, CTLA4Ig-treated cardiac allograft at day 62 showsextensive lymphocytic infiltration, interstitial fibrosis, and severecoronary arterial intimal thickening and fibrosis consistent withchronic rejection.

[0117] In FIG. 2C, MR1-treated cardiac allograft at day 62 demonstratesless lymphocytic infiltration and interstitial fibrosis, but severecoronary vasculopathy characteristic of chronic rejection.

[0118] In FIG. 2D, CTLA4Ig/MR1-treated cardiac allografts at day 58, inmarked contrast, were remarkably free from lymphocytic infiltration,fibrosis, and most significantly, coronary arterial intimal lesions. Theparenchyma and blood vessels of these grafts were virtuallyindistinguishable from normal untransplanted BALB/c hearts.

[0119] In FIG. 2E, normal untransplanted BALB/c hearts are shown.

[0120] Similar histologic results were obtained from three allografts ineach experimental group. C3H/HeJ (H-2^(k)) recipients treated withCTLA4Ig alone, MR1 alone, or CTLA4Ig/MR1, all showed prolonged survivalof BALB/c (H-2^(d)) cardiac allografts when compared to untreatedcontrols (FIG. 2A). However, when examined histologically at 58-62 dayspost-transplant marked differences were apparent.

[0121] Allografts from CTLA4Ig-treated recipients showed extensivelymphocytic infiltration, interstitial fibrosis, and severe coronaryarterial intimal thickening and fibrosis consistent with chronicrejection (FIG. 2B). While the MR1-treated allograft demonstrated lesslymphocytic infiltration and interstitial fibrosis, these grafts alsohad severe coronary vasculopathy characteristic of chronic rejection(FIG. 2C).

[0122] In marked contrast, the allograft from CTLA4Ig/MR1 treatedrecipients were remarkably free from lymphocytic infiltration, fibrosis,and most significantly, coronary arterial intimal lesions (FIG. 2D). Infact, the parenchyma and blood vessels of these grafts were virtuallyindistinguishable from those found in normal BALB/c hearts (FIG. 2E).

EXAMPLE 3

[0123] This example shows blockade of T cell cytokine and costimulatorymolecule transcript expression.

[0124] Method

[0125] At 8 days after transplantation, the cardiac grafts were removedand total RNA was prepared from tissues using TRIzol Reagent (GIBCO BRL,Gaithersburg, Md.). CDNA was synthesized using 5 μg of total RNAtemplate with a Superscript Preamplification System (GIBCO BRL,Gaithersburg, Md.) in a final volume of 20 μl. PCR reactions werecarried out. PCR products were visualized on ethidium bromide stained 1%agarose (BIO-RAD, Hercules, Calif.), 2% NuSieve GTG agarose (FMCBioProducts, Rockland, Me.) gels. Gel images were stored using a UVP GelDocumentation System 5000. Band intensity was quantified using Gelreaderanalysis software (National Center for Supercomputing Applications,Urbana, Ill.).

[0126] In FIG. 3A, intragraft expression of immune mediator transcriptswas assessed using RT-PCR in untreated, MR1-treated, CTLA4Ig treated,and MR1/CTLA4Ig treated cardiac allografts.

[0127] Three allografts from each treatment group and the control groupwere analyzed at 8 days post-transplant. Normal heart tissue (N) and asyngeneic heart graft (S) at 8 days after transplantation were includedfor comparison.

[0128] In FIG. 3B, graphical representation of the mean PCR product bandintensities±standard deviation are shown.

[0129] Discussion

[0130] No consistent differences in the expression of T cell cytokinetranscripts for IL-2, IL-4, IL-10, and IFNγ or costimulatory moleculetranscripts (B7-1, and B7-2) were detectable between the controlallografts (FIG. 3A, untreated) and MR1-treated allografts (FIG. 3A),whereas CTLA4Ig partially inhibited expression of IL-4 transcripts.

[0131] Allografts from CTLA4Ig/MR1-treated recipients showed a strikingdecrease in the expression of both Th1 cytokine (IL-2 and IFNγ) and Th2cytokine (IL-4, and IL-10) transcripts. However, intragraft B7-1 andB7-2 costimulatory molecule transcripts were only modestly reduced inrecipients treated with CTLA4Ig/MR1.

[0132] PCR reactions using template prepared without reversetranscriptase yielded no products, even for the intron-less GADPH gene(FIG. 3A, GADPH, NO RT), confirming the absence of contaminating genomicDNA.

[0133] Intragraft B7-1 and B7-2 costimulatory molecule transcripts wereonly modestly reduced in recipients treated with CTLA4Ig/MR1, (FIG. 3)suggesting that CD28/B7-independent or CD40/gp39-independent factors,such as GMCSF (Larsen, C. P., et al. J Immunol 152, 5208-5219 (1994)),may be important regulators of intragraft B7 expression. Thus,MR1-mediated blockade of the CD40 pathway not only inhibits T cellcognate help for effector APC's, but enhances the ability of CTLA4Ig toinhibit T cell activation transcript expression within allografts. Thesedata are consistent with those from our studies in vitro which indicatethat while MR1 alone has only a modest negative effect on cellularproliferation in allogeneic mixed leukocyte reactions, it potentiatesthe inhibitory effects of suboptimal concentrations of CTLA4Ig.

EXAMPLE 4

[0134] This example demonstrates prolongation of murine skin allograftsurvival using C3H/HeJ mice which received full thickness skinallografts from BALB/c mice.

[0135] Method

[0136] Segments of either full thickness tail or ear skin ofapproximately 1 cm square were grafted on to the posterior-lateralthoracic wall of recipient mice and secured in place with acircumferential Bandaid®. The grafts were then followed by daily visualinspection. Rejection was defined as the complete loss of visibleepidermal graft tissue. Treatment protocols for MR1 and CTLA4Ig were asdetailed for heart transplant recipients in FIG. 1. CyA (Sandoz, EastHanover, N.J.) at a concentration of 50 mg/ml was administered at a rateof 0.5 μl/hr (˜20 mg/kg/day) for 14 days via an osmotic pump (AlzetModel No. 2002, Alza, Palo Alto, Calif.) which was implantedsubcutaneously in the dorsal region of the recipient at the time of skingrafting and removed at 21 days after transplant (Pereira, G. M.,Miller, J. F. & Shevach, E. M. J Immunol 144, 2109-2116 (1990)). Aftersacrifice, the skin graft was excised, formalin fixed and embedded inparaffin. Tissue sections (5 μm) were stained with hematoxylin-eosin.

[0137] In FIG. 4A, C3H/HeJ recipients treated with either MR1 alone(MST=13 days, n=5) or CTLA4Ig alone (MST=12 days, n=7) rejected fullyMHC-disparate BALB/c skin grafts at the same rate as an untreatedcontrol group (MST=13 days, n=5). In contrast, when MR1 and CTLA4Ig wereadministered together in the perioperative period, the allograftsenjoyed markedly prolonged survival (MST>50, n=15).

[0138] In FIG. 4B, mice treated with CyA alone (MST=30 days, n=4), CyAplus CTLA4Ig (MST=30 days, n=5), or CyA and MR1 (MST=32 days, n=4) alldisplayed similar modestly prolonged skin graft survival. Surprisingly,the salutary effect of CTLA4Ig/MR1 on skin graft survival was abolishedby concomitant cyclosporine administration (MST=34 days, n=4).

[0139] in FIG. 4C, C3H recipients of BALB/c skin grafts were not treated(MST 10d, n=3), or treated with MR1 (MST 13d, n=3), YTS191.1 (MST 14d,n=6), YTS191.1 and MR1 (MST 16d, n=6), YTS191.1 and CTLA4Ig (MST 19d,n=5), or CTLA4Ig and MR1 (MST >50d, n=22). Thus far, >53 mice have beentreated with CTLA4Ig/MR1. Of these, 2 died on days 13 and 21. All othershave remained healthy throughout the experiments without signs of weightloss, infection, or malignancy. Healthy appearance of a BALB/c skingraft on an CTLA4Ig/MR1 treated C3H/HeJ recipient at 50 days aftertransplant (FIG. 4D), contrasts sharply with a control allograftundergoing rejection (FIG. 4E). On hematoxylin-eosin stained sectionsthe accepted graft at 100 days after transplant demonstrated wellpreserved epidermis, hair follicles and adnexal structures (FIG. 4F),which is in contrast to a BALB/c skin graft on an untreated C3H/HeJrecipient 8 days after transplant which shows an extensive lymphocyticinfiltrate (FIG. 4G).

[0140] Discussion

[0141] The effects of CTLA4Ig and MR1, alone and in combination onprimary skin allograft survival in mice were tested (FIG. 4). Forcomparison, recipients were also treated with CyA alone or CyA combinedwith either CTLA4Ig or MR1. C3H/HeJ recipients treated with either MR1alone or CTLA4Ig alone rejected fully MHC-disparate BALB/c skin graftsat the same rate as untreated controls (FIG. 4A).

[0142] Mice treated with CyA alone, CyA plus CTLA4Ig, or CyA and MR1 alldisplayed modestly prolonged skin graft survival (FIG. 4B). However, allof these allografts were ultimately rejected with no apparent effectsbetween either drug and CyA.

[0143] As a rigorous test of the ability of CD40/CD28 blockade tointerrupt alloimmune responses, we studied the effects of perioperativetreatment with CTLA4Ig and MR1, alone and in combination on primary skinallograft survival in mice. For comparison, recipients were also treatedwith CyA, an anti-CD4 mAb YTS191.1, or with either of these agentscombined with CTLA4Ig or MR1 (FIGS. 4B and 4C). C3H/HeJ recipientstreated with either MR1, CTLA4Ig, or YTS191.1 alone rejected fullyMHC-disparate BALB/c skin grafts at essentially the same rate asuntreated controls (FIGS. 4B and 4C). Mice treated with YTS191.1 andMR1, YTS191.1 and CTLA4Ig, CyA alone, CyA plus CTLA4Ig, or CyA and MR1displayed modestly prolonged skin graft survival (FIGS. 4B and 4C).However, all of these allografts were ultimately rejected.

[0144] In contrast, on recipients treated with both MR1 and CTLA4Ig inthe perioperative period, the skin allografts demonstrated markedlyprolonged survival. Visual examination of these allografts at 50 daysafter transplantation showed the grafts to be healthy in appearance,well vascularized, supple, and bearing short white hair (FIG. 4D).Histologically, the accepted grafts demonstrated well preservedepidermis, hair follicles and adnexal structures (FIG. 4F).Surprisingly, the salutary effect of CTLA4Ig/MR1 on skin graft survivalwas abolished by concomitant cyclosporine administration (FIG. 4B).

[0145] The remarkable potency of this effect was most clearly evident inthe primary skin allograft model. Neither CTLA4Ig or MR1 alone or withCyA significantly prolonged skin allograft survival. Only thecombination of CTLA4Ig and MR1 produced >50 day survival of fully-MHCmismatched skin allografts. Similar prolongation in this stringent testof inhibition of the alloimmune response has previously only beenobserved using vigorous cytoablative and/or hematopoieticchimerism-based strategies (Mayumi, H. & Good, R. A. J Exp Med 169,213-238 (1990); Ildstad, S. T. & Sachs, D. H., Nature 307, 168-170(1984); Ilstad, S. T., et al. J Exp Med 162, 231-44 (1985); Cobbold, S.P., Martin, G., et al. Nature 323, 164-166 (1986); Qin, S., et al.Science 259, 974-977 (1993)).

EXAMPLE 5

[0146] To explore the effect of blockade of the CD28 and CD40 pathwayson T cell proliferation, we studied the primary allogeneic mixedleukocyte reaction using T cells from both Ie^(k)-restricted pigeoncytochrome c-reactive (pcc-TCRTg) and L^(d)-alloreactive (2C) T cellreceptor transgenic mice (REF HED and LOH). CTLA4Ig, a fusion proteinwhich binds to the ligands for CD28 and its homologue CTLA4, effectivelyinhibited proliferation of all three T cell populations (FIG. 5A).

[0147] In contrast, blockade of the CD40 pathway with the hamsteranti-gp39 mAb, MR1, modestly (˜50%) inhibited the proliferation ofC3H/HeJ T cells responding to BALB/c dendritic cells, dramaticallyinhibited (≧85%) pcc-TCRTg T cells to reacting to cytochrome c, but hadnegligible effects on the proliferation of 2C T cells responding toL^(d)-bearing BALB/c dendritic cells (FIG. 5A).

[0148] Furthermore, simultaneous blockade with these agentscooperatively inhibited T cell proliferation in allogeneic mixedleukocyte reactions and pcc-TCRTg T cells, whereas MR-1 had no effect orslightly augmented the proliferation of 2C T cells when combined withCTLA4Ig (FIG. 5A). These results indicate that not all T cells aredependent on CD40 signals for clonal expansion and may explain theinability of CD40 blockade to completely inhibit allograft rejection.

[0149] The effect of CD28 and CD40 blockade on T cell responses in vivowas assessed in C3H/HeJ (H-2^(k), MMTV-7⁻) immunized with DBA/2(H-2^(d),MMTV-7⁺) splenocytes in their foot pads. Five days after immunizationthe draining popliteal lymph nodes in control mice treated with humanIgG demonstrated a 4-6 fold increase in weight (FIG. 5B). This wasaccompanied by a 30 fold expansion in the number of MMTV-7superantigen-reactive Vβ⁺CD⁺4 T cells and a >90 fold increase in thenumber of Vβ⁺CD⁺T cell blasts within the popliteal lymph node. Alone,CTLA4Ig or MR1 partially inhibited these responses. In contrast, thecombination of CTLA4Ig/MR1 essentially ablated the increase in lymphnode size and the expansion and blastogenesis of Vβ⁺CD4⁺T cells (FIG.5B).

[0150] These data show that simultaneous blockade of the CD28 and CD40pathways inhibit complex T-dependent immune responses in vitro and invivo.

EXAMPLE 6

[0151] This example shows that simultaneous blockade of the CD28 andCD40 pathways produces marked inhibition of both the cellular andantibody response to xenoantigen and long-term acceptance of xenogeneic(rat to mouse) cardiac and skin grafts without the need for acytoablative conditioning therapy.

[0152] Methods

[0153] LYMPH NODE ASSAY. Male C3H (Jackson Laboratory, Bar Harbor, Me.)mice were immunized with 2×10⁶ male Sprague-Dawley (Harlan,Indianapolis, Ind.) irradiated (2000 RADS) rat splenocytes in 50 μl ofsterile normal saline in the left foot pad and 50 μl of sterile normalsaline in the right foot pad and then treated intraperitoneally (i.p.)with MR1 (500 μg), CTLA4-Ig (500 μg) or both reagents (500 μg each) ondays 0, 2 and 4. On day 5, the popliteal lymph nodes were removed,weighed and then teased apart, washed, before resuspension in 600 μl ofRPMI 1640 with 10% FBS (Mediatech, Herndon, Va.). Each resuspended nodewas then divided into four equal aliquots (150 μl each). Three of thealiquots were plated into a 96 well plate. ³H-thymidine (1 μCi/well)(Amersham, Arlington Heights, Ill.) incorporation was measured after 24hours incubation at 37° C. The results for each individual animaltherefore represent the mean of the 3 wells per node. The fourth aliquotwas incubated for 24 hours at 37° C. and served as the source ofsupernatant for cytokine analysis with ELISA. Each point on all of thegraphs represents the mean±standard deviation of 5 mice per group. Theexperiment was repeated with similar results.

[0154] CYTOKINE ELISA. Sandwich ELISA was performed using pairedantibodies {anti-IL-2, anti-IFN-gamma, anti-IL-2 biotin, anti-IL-4biotin, anti-IFN-gamma biotin (Pharmingen, San Diego, Calif.), anti IL4(kind gift from Peter Jensen)} and streptavidin-HRP (Pierce, Rockford,Ill.). Colorimetric detection was assayed using TMB substrate (Pierce).Data were collected using a SpectraMax plate reader and plotted asabsorbance (370 nm)+/−sem. Standard curves for each cytokine weregenerated using recombinant cytokine (rIL2, Boehringer Mannheim,Indianapolis, Ind.; rIL4, R&D Systems, Minneapolis, Minn.; andrIFN-gamma, Biosource International, Camarillo, Calif.).

[0155] MICE. Male C3H/HeJ (H-2 ^(k)) and DBA/2 (H-2^(d)) mice andSprague-Dawley rats were purchased from The Jackson Laboratory (BarHarbor, Me.) and used at 8-12 weeks of age.

[0156] CARDIAC TRANSPLANTATION. C3H/HeJ or DBA mice were transplantedwith primarily vascularized Sprague-Dawley rat heart xenografts andmonitored for rejection as described in Larsen C. P., Alexander D. Z.,Hollenbaugh D., et al., Transplantation, 61(1):4-9 (1996) and Corry R.J., Winn H. J., Russell P. S., Transplantation, 16(4):343-350 (1973).Recipients were treated with 500 mg CTLA4-Ig combined with 500 mg MR1 ondays 0, 2, 4 and 6. Control groups included recipients treated withCTLA4-Ig alone, MR1 alone or Human Ig. Paraffin embedded tissue sections(5 μm) were stained with Masson's Trichrome or hematoxylin-eosin.Histologic specimens were reviewed by a cardiac transplant pathologist(KJW) blinded to the treatment modality.

[0157] SKIN TRANSPLANTATION. Full thickness skin grafts (˜1 cm²) fromSprague-Dawley rats were transplanted on the dorsal thorax of C3Hrecipient mice and survival followed by daily visual inspection.Rejection was defined as the complete loss of visible epidermal grafttissue. Control groups included recipients treated with: CTLA4-Ig alone;MR1 alone; and Human Ig. Two additional mice in each experimental groupwere sacrificed at 20 days post transplant for histologic analysis.

[0158] XENOANTIBODY ASSAY. Serum was collected via tail bleed fromanesthetized animals. Single cell suspensions from lymph nodes of aSprague-Dawley rat were used as target cells, and incubated withrecipient mouse serum for 20 minutes at 4° C. The cells were washed andIgG xenoantibodies were detected with donkey anti-mouse IgG Biotin(Jackson ImmunoResearch, West Grove, Pa.) followed by streptavidin-PE(Southern Biotech, Birmingham, Ala.). Cells were analyzed on aBecton-Dickinson FACscan using Cellquest Software.

[0159] Results

[0160] As an initial approach to determine the effects of CD28 and CD40blockade on responses to xenoantigenic challenge in vivo, we used thepopliteal lymph node assay as described in Larsen C. P., Elwood E. T.,Alexander D. Z., et al., Nature, 381:434-438 (1996). C3H/HeJ (H-2^(k))mice were injected with irradiated Sprague-Dawley (SD) rat splenocytes.Five days after foot pad immunization, the draining popliteal lymphnodes in control mice treated with human IgG demonstrated a 5.2 foldincrease in weight relative to the contralateral node which wasinoculated with sterile saline (FIG. 6A). CTLA4-Ig partially inhibitednodal expansion. Similarly MR1 partially inhibited this response. Incontrast, the combination of CTLA4-Ig/MR1 essentially ablatedxenoantigen-induced lymph node expansion (FIG. 6A).

[0161] We then compared the ex vivo proliferation of lymph node cellsfrom the different groups of xenoantigen-primed mice. While eithertreatment alone only partially blocked proliferation (FIG. 6B), thecombination of CTLA4-Ig/MR1 essentially ablated the proliferativeresponse (302+/−235 CPM for the combination versus 143+/−145 for anormal unstimulated node). Furthermore, the combination therapy markedlysuppressed Th1 cytokines (IL-2 and IFNg) to the level of normalunstimulated cells (FIGS. 6C and 6D). Levels of the Th2 cytokine IL4were below the level of detection in all samples.

[0162] It is important to note that this potent immune modulation is notthe result of cellular deletion. Flow cytometric analysis of theperipheral blood of treated mice showed no depletion of CD4+ or CD8+ Tcells, B cells, or NK cells. These data are the result of an individualanalysis of 3 mice per group treated with either CTLA4-Ig or MR1 aloneor the combination of these agents on days 0, 2, 4, and 6 as describedin the methods section. Peripheral blood was collected by tail bleed ondays 6 and 20.

[0163] The results of the lymph node assays suggested that simultaneousblockade of the B7/CD28 and CD40/gp39 pathways would inhibit xenograftrejection. To explore this hypothesis we studied a vascularized cardiacxenograft model using Sprague-Dawley rats as donors and C3H/HeJ mice asrecipients. Treatment with either CTLA4-Ig (MST=33 days) or MR1 (MST=51days) alone prolonged xenograft survival when compared to untreatedcontrols (MST=6 days) (FIG. 7A). However, CTLA4-Ig/MR1 in combinationmarkedly prolonged survival (MST=104.5 days).

[0164] When examined histologically at 20 days post-transplant,xenografts treated with either CTLA4-Ig alone (FIG. 7C) or MR1 alone(FIG. 7D) showed heavy lymphocytic infiltration with evidence of myocytedestruction and vasculopathy consistent with moderate to severe cellularrejection. In sharp contrast, the xenografts from CTLA4-Ig/MR1 treatedrecipients were essentially free from lymphocytic infiltration,interstitial fibrosis, and coronary arterial intimal lesions (FIG. 7F).CTLA4-Ig/MR1-treated cardiac xenografts demonstrated excellentpreservation of both myocytes and vascular structures at day 122 (FIG.7G) Untreated xenografts showed widespread tissue destruction at day 6(FIG. 7B). A normal untransplanted Sprague-Dawley rat heart is shown inFIG. 7E.

[0165] As a more stringent test of the ability of CD40/CD28 blockade toinhibit xenogeneic immune responses, we studied the effects of shortterm CD28 and /or CD40 blockade, on primary skin xenograft survival inmice.

[0166] C3H recipients treated with either MR1 (MST=11.5 days n=4) orCTLA4-Ig (MST=12 days n=4) alone rejected full thickness skin graftsfrom Sprague-Dawley rats at the same rate as untreated controls(untreated controls MST=11.5 days n=8) (FIG. 8A). In contrast, the skinxenografts on recipients treated with simultaneous MR1 and CTLA4-Ig inthe perioperative period, demonstrated markedly prolonged survival(MST=53 days n=25) (FIG. 8A). A total of 25 mice received xenografts andtreatment with CTLA4-Ig/MR1. With the exception of one mouse that diedon day 4, all others have remained healthy throughout the experimentswithout signs of weight loss, infection, or malignancy.

[0167] Chronic treatment (beginning after the standard 4 dose regimen)with either the CTLA4-Ig/MR1 combination (500 μg of both agents weeklyuntil day 100 or rejection, whichever came first) or MR1 (500 μg of MR1weekly until day 100 or rejection) resulted in no significant change inxenograft survival (FIG. 8B).

[0168] Xenograft survival after simultaneous MR1/CTLA4-Ig therapy was52% and 21% at 50 and 100 days respectively. The xenografts surviving at50 (FIG. 8E) and 100 (FIG. 8C) days after transplantation were healthyin appearance and demonstrated well preserved histologic architecture.In untreated controls without the combination therapy, rejection wasprompt and these xenografts showed marked inflammatory infiltrates(FIGS. 8D and 8F).

[0169] Similar prolongation of Sprague-Dawley skin xenografts was alsoobserved in DBA[H-2 ^(d)]) recipients (untreated controls MST=14 days(n=5) versus CTLA4-Ig/MR1 MST=86 days n=5), suggesting that the potenteffect of the combination treatment is not limited to a single recipientmouse strain.

[0170] The progressive loss of skin xenografts between 25 and 75 dayspost-transplant suggested that late graft failure might be due tosubtherapeutic concentrations of CTLA4-Ig and/or MR1. To address thispossibility, after the standard four-dose combination regimen mice weretreated weekly with either the CTLA4-Ig/MR1 combination or MR1 alone for100 days or until graft loss occurred. Neither of these chronic therapystrategies appreciably improved skin xenograft survival, suggesting thatgraft failure in CTLA4-Ig/MR1 treated mice results from factors otherthan inadequate drug titers and that alternate pathways not completelyinhibited by CTLA4-Ig/MR1 may be important in sub-acute xenograft loss.

[0171] In addition to cell-mediated effector mechanisms, evokedxenoantibody responses may play an important role in acceleratedvascular rejection of concordant xenografts, Aksentijevich I., Sachs D.H., Sykes M., Transplantation, 53(5):1108-14 (1992). To test the effectof simultaneous blockade on evoked xenoantibody responses, serum samplesfrom C3H/HeJ mice were analyzed for anti-rat antibodies at 55 days afterreceiving a Sprague-Dawley rat skin graft (FIG. 9A) and at 20 days afterreceiving a Sprague-Dawley rat heart graft (FIG. 9B). Treatment witheither CTLA4-Ig or MR1 alone decreased the IgG antibody response,whereas the simultaneous combination CD28/CD40 blockade essentiallyeliminated the evoked antibody response to rat xenoantigens. Thus,inhibition of both T cell activation and antibody production could befunctionally important in xenograft survival after simultaneous blockadeof the B7/CD28 and CD40/gp39 pathways.

[0172] Discussion

[0173] Combined blockade of the CD28 and CD40 pathways markedly inhibitsthe immune response to xenoantigen. The potency of this combinationtherapy was particularly demonstrated in the primary skin xenograftmodel. Neither agent alone prolonged skin xenograft survival, while, incontrast, the simultaneous combination of CTLA4-Ig and MR1 cooperativelyinhibited xenograft rejection. The uniqueness of the findings resides inthe stringency of the skin graft model, as CTLA4-Ig alone has previouslybeen shown to prolong the survival of xenogeneic pancreatic islets in amouse model, Lenschow D., Zeng Y., Thistlethwaite J., et al., Science,257:789-792 (1992). Long-term survival of xenogenic skin grafts havepreviously only been observed using vigorous cytoablative and/orhematopoeitic chimerism-based strategies, Ildstad S. T., Sachs D. H.,Nature, 307:168-170 (1984), Zhao Y., Swenson K., Sergio J., Arn J. S.,Sachs D. H., Sykes M., Nat. Med., 2(11):1211-1216 (1996), Mayumi H.,Good R. A., J. Exp. Med., 169(1):213-238 (1990), Cobbold S. P., MartinG., Zin S., Waldman H., Nature, 323:164-166 (1986), Qin S., Cobbold S.,Benjamin R., Waldmann H., J. Exp. Med., 169:779-794 (1989).

[0174] The observation that simultaneous CD28/CD40 blockade candramatically prolong xenograft survival suggests that both the antibodyand cell mediated mechanisms for destruction of the xenograft may beeffectively inhibited by this strategy. While the etiology of acutevacular xenograft rejection remains to be completely defined, there isevidence that it is caused, at least in part, by the development ofxenoreactive antibodies (Cotterell A. H., Collins B. H., Parker W.,Harland R. C., Platt J. L, Transplantation, 60(8):861-868 (1995)). Therapid destruction of untreated control cardiac xenografts in our model,in the absence of a cellular infiltrate, suggests a role for antibodymediated rejection. This observation and those of others (AksentijevichI., Sachs D. H., Sykes M., Transplantation, 53(5):1108-14 (1992)),combined with the documented dramatic inhibition of the evokedxenoantibody response after blockade of the CD28 and CD40 pathways(FIGS. 9A and 9B), supports the hypothesis that xenoresponses may besufficiently controlled by inhibition of these pathways to permit thedevelopment of non-cytoablative strategies for xenotransplantation indiscordant species combinations.

[0175] While combined blockade of the CD28 and CD40 pathways markedlyinhibited the xenograft rejection response, this blockade did notachieve uniform indefinite cardiac or skin graft survival in ourexperimental system. The observation that prolonged treatment did notimprove graft survival was surprising. This suggests that inadequateblockade of these pathways is not the cause for “late” graft failure andraises the possibility that alternate pathways such as NK cells or othercells, which do not require CD28/CD40 co-stimulation, may promote latexenograft rejection. Suggestive evidence for the contribution of NKcells to xenograft rejection support this possibility, Zhao Y., SwensonK., Sergio J., Arn J. S., Sachs D. H., Sykes M., Nat. Med.,2(11):1211-1216 (1996), Malyguine A. M., Saadi S., Platt J. L., DawsonJ. R., Transplantation, 61(1):161-164 (1996). In addition, we have shownthat the inhibition of the rejection of concordant heart and skinxenografts by the simultaneous blockade of the CD40 and CD28 pathways isassociated with the prevention of the “late” development of the evokedxenoreactive antibody responses (FIGS. 9A and 9B), we have not excludedthe possibility that the development of an antibody response may beassociated with delayed graft loss.

[0176] The ability of combined CTLA4-Ig/MR1 treatment to block thedevelopment of transplant vasculopathy in cardiac xenografts and prolongskin xenografts is of significant clinical relevance. The refinement oftechniques to inhibit the effect of natural preformed xenoreactiveantibodies combined with further study of CD28 and CD40 pathway blockadepromises the possibility of effective new strategies to facilitateclinical xenograft transplantation.

What is claimed is:
 1. A method for inhibiting rejection of atransplanted tissue comprising: a) contacting a B7-positive cell with afirst soluble ligand which recognizes and binds the B7 antigen, and b)contacting a gp39-positive cell with a second soluble ligand whichrecognizes and binds the gp39 antigen, the binding of the B7-positivecell to the first soluble ligand thereby blocking the reaction of the B7antigen with endogenous CTLA4 or CD28 and the binding of the gp39antigen to the second soluble ligand thereby blocking the reaction ofgp39 antigen with endogenous CD40, the blockage thereby inhibiting theimmune response.
 2. A method for inhibiting an immune response mediatedby CTLA4-, CD28-, and gp39-positive cell interactions with B7- andCD40-positive cells in a subject comprising administering to the subjecta first soluble ligand which recognizes and binds the B7 antigen and asecond soluble ligand which recognizes and binds the gp39 antigen, suchbinding thereby inhibiting the immune response mediated by CTLA4-,CD28-, and gp39-positive cell interactions with B7-positive cells andgp39-positive cells.
 3. A method for inhibiting transplant rejection ina subject comprising administering to the subject an effective amount ofa combination of a first soluble ligand which recognizes and binds theB7 antigen on B7-positive cells and a second soluble ligand whichrecognizes and binds the gp39 antigen on gp39-positive cells, thebinding of B7-positive cells with the first soluble ligand andgp39-positive cells with the second soluble ligand thereby disruptingendogenous CTLA4-, CD28-, and gp39-positive cell interactions withB7-positive cells and gp39-positive cells so that transplant rejectionis inhibited.
 4. The method of claim 1, 2, or 3, wherein the firstsoluble ligand is a recombinant binding molecule having at least aportion of the extracellular domain of CTLA4.
 5. The method of claim 4,wherein the ligand is CTLA4Ig fusion protein.
 6. The method of claim 4,wherein the ligand is a CD28 Ig/CTLA4Ig fusion protein hybrid.
 7. Themethod of claim 5, wherein the CTLA4Ig fusion protein is CTLA4Igdesignated ATCC
 68629. 8. The method of claim 1, 2, or 3, wherein thefirst soluble ligand is a monoclonal antibody reactive with B7 antigen.9. The method of claim 8, wherein the antibody is anti-BB1 monoclonalantibody.
 10. The method of claim 6, wherein the ligand is a CD28Ig/CTLA4Ig fusion protein hybrid having a first amino acid sequencecorresponding to a portion of the extracellular domain of CD28 receptorfused to a second amino acid sequence corresponding to a portion of theextracellular domain of CTLA4 receptor and a third amino acid sequencecorresponding to the hinge, CH2 and CH3 regions of human immunoglobulinCg1.
 11. The method of claim 1, 2, or 3, wherein the second solubleligand for the gp39 antigen is a monoclonal antibody reactive with thegp39 antigen.
 12. The method of claim 11, wherein the antibody is MR1monoclonal antibody.
 13. The method of claim 4, wherein theextracellular portion of CTLA4 is joined to a non-CTLA4 proteinsequence.
 14. The method of claim 13, wherein the non-CTLA4 proteinsequence is at least a portion of an immunoglobulin molecule.
 15. Themethod of claim 2 or 3, wherein the subject is an animal subject. 16.The method of claim 15, wherein the animal subject is a human.
 17. Amethod for inhibiting an immune response comprising: a) preventing anendogenous antigen on a cell selected from the group consisting of gp39and CD40 from binding its endogenous ligand; and b) preventing anendogenous antigen on a cell selected from the group consisting ofCTLA4, CD28, and B7 from binding its endogenous ligand, the preventionof such antigens from binding their ligand thereby blocking twoindependent cell signals and inhibiting the immune response.
 18. Themethod of claim 17, wherein: a) the step of preventing the endogenousgp39 antigen from binding its endogenous ligand comprises contacting agp39-positive cell with a soluble ligand which recognizes and binds thegp39 antigen, b) the step of preventing the endogenous CTLA4 antigenfrom binding its endogenous ligand comprises contacting a B7-positivecell with a soluble ligand which recognizes and binds the B7 antigen,the binding of the gp39-positive cell to its soluble ligand of step (a)thereby blocking the reaction of endogenous gp39 antigen with endogenousCD40, the binding of the B7-positive cell to its soluble ligand of step(b) thereby blocking the reaction of the endogenous B7 antigen withendogenous CTLA4, the blockage thereby inhibiting the immune response.19. The method of claim 18, wherein the soluble ligand of step (a) is amonoclonal antibody reactive with the gp39 antigen.
 20. The method ofclaim 18, wherein the soluble ligand of step (b) is CTLA4Ig.
 21. Themethod of claim 17, wherein: a) the step of preventing the endogenousCD40 antigen from binding its endogenous ligand comprises contacting aCD40-positive cell with a soluble ligand which recognizes and binds theCD40 antigen, b) the step of preventing the endogenous CTLA4 antigenfrom binding its endogenous ligand comprises contacting a B7-positivecell with a soluble ligand which recognizes and binds the B7 antigen,the binding of the CD40-positive cell to its soluble ligand of step (a)thereby blocking the reaction of endogenous CD40 antigen with endogenousgp39, the binding of the B7-positive cell to its soluble ligand of step(b) thereby blocking the reaction of the B7 antigen with endogenousCTLA4, the blockage thereby inhibiting the immune response.
 22. Themethod of claim 21, wherein the soluble ligand of step (a) is amonoclonal antibody directed against CD40.
 23. The method of claim 21,wherein the soluble ligand of step (b) is CTLA4Ig.
 24. The method ofclaim 17, wherein: a) the step of preventing the endogenous gp39 antigenfrom binding its endogenous ligand comprises contacting a gp39-positivecell with a soluble ligand which recognizes and binds the gp39 antigen,b) the step of preventing the endogenous CD28 antigen from binding itsendogenous ligand comprises contacting a B7-positive cell with a solubleligand which recognizes and binds the B7 antigen, the binding of thegp39-positive cell to its soluble ligand of step (a) thereby blockingthe reaction of gp39 antigen with endogenous CD40, the binding of theB7-positive cell to its soluble ligand of step (b) thereby blocking thereaction of the B7 antigen with endogenous CD28, the blockage therebyinhibiting the immune response.
 25. The method of claim 24, wherein thesoluble ligand of step (a) is a monoclonal antibody reactive with thegp39 antigen.
 26. The method of claim 24, wherein the soluble ligand ofstep (b) is CTLA4Ig.
 27. The method of claim 17, wherein: a) the step ofpreventing the endogenous CD40 antigen from binding its endogenousligand comprises contacting a CD40-positive cell with a soluble ligandwhich recognizes and binds the CD40 antigen, b) the step of preventingthe endogenous B7 antigen from binding its endogenous ligand comprisescontacting a CD28-positive cell with a soluble ligand which recognizesand binds the CD28 antigen, the binding of the CD40-positive cell to thesoluble ligand of step (a) thereby blocking the reaction of CD40 antigenwith endogenous gp39, the binding of the CD28-positive cell to thesoluble ligand of step (b) thereby blocking the reaction of the CD28antigen with endogenous B7, the blockage thereby inhibiting the immuneresponse.
 28. The method of claim 27, wherein the soluble ligand of step(a) is a monoclonal antibody directed against CD40.
 29. The method ofclaim 27, wherein the soluble ligand of step (b) is a monoclonalantibody directed against CD28.
 30. The method of claim 17, wherein: a)the step of preventing the endogenous CD40 antigen from binding itsendogenous ligand comprises contacting a CD40-positive cell with asoluble ligand which recognizes and binds the CD40 antigen, b) the stepof preventing the endogenous B7 antigen from binding its endogenousligand comprises contacting a CTLA4-positive cell with a soluble ligandwhich recognizes and binds the CTLA4 antigen, the binding of theCD40-positive cell to the soluble ligand of step (a) thereby blockingthe reaction of CD40 antigen with endogenous gp39, the binding of theCTLA4- or CD28-positive cell to the soluble ligand of step (b) therebyblocking the reaction of the CTLA4 antigen with endogenous B7, theblockage thereby inhibiting the immune response.
 31. The method of claim30, wherein the soluble ligand of step (a) is sgp39.
 32. The method ofclaim 30, wherein the soluble ligand of step (b) is monoclonal antibodydirected against CTLA4.
 33. The method of claim 17, wherein: a) the stepof preventing the endogenous CD40 antigen from binding its endogenousligand comprises contacting a gp39-positive cell with a soluble ligandwhich recognizes and binds the gp39 antigen, b) the step of preventingthe endogenous B7 antigen from binding its endogenous ligand comprisescontacting a CD28-positive cell with a soluble ligand which recognizesand binds the CD28 antigen, the binding of the gp40-positive cell to thesoluble ligand of step (a) thereby blocking the reaction of gp39 antigenwith endogenous CD40, the binding of the CD28-positive cell to thesoluble ligand of step (b) thereby blocking the reaction of the CD28antigen with endogenous B7, the blockage thereby inhibiting the immuneresponse.
 34. The method of claim 33, wherein the soluble ligand of step(a) is a monoclonal antibody reactive with the gp39 antigen.
 35. Themethod of claim 33, wherein the soluble ligand of step (b) is monoclonalantibody directed against CD28.
 36. The method of claim 17, wherein: a)the step of preventing the endogenous CD40 antigen from binding itsendogenous ligand comprises contacting a gp39-positive cell with asoluble ligand which recognizes and binds the gp39 antigen, b) the stepof preventing the endogenous B7 antigen from binding its endogenousligand comprises contacting a CTLA4-positive cell with a soluble ligandwhich recognizes and binds the CTLA4 antigen, the binding of theCD40-positive cell to the soluble ligand of step (a) thereby blockingthe reaction of gp39 antigen with endogenous CD40, the binding of theCTLA4-positive cell to the soluble ligand of step (b) thereby blockingthe reaction of the CTLA4 antigen with endogenous B7, the blockagethereby inhibiting the immune response.
 37. The method of claim 36,wherein the soluble ligand of step (a) is a monoclonal antibody reactivewith the gp39 antigen.
 38. The method of claim 36, wherein the solubleligand of step (b) is monoclonal antibody directed against CTLA4.
 39. Apharmaceutical composition useful to inhibit an immune responsecomprising a pharmaceutically effective amount of a soluble ligand whichrecognizes and binds a B7 antigen and an acceptable carrier.
 40. Apharmaceutical composition useful to inhibit an immune responsecomprising a pharmaceutically effective amount of a soluble ligand whichrecognizes and binds a CD28 antigen and an acceptable carrier.
 41. Apharmaceutical composition useful to inhibit an immune responsecomprising a pharmaceutically effective amount of a soluble ligand whichrecognizes and binds a CTLA4 antigen and an acceptable carrier.
 42. Apharmaceutical composition useful to inhibit an immune responsecomprising a pharmaceutically effective amount of a soluble ligand whichrecognizes and binds a gp39 antigen and an acceptable carrier.
 43. Apharmaceutical composition useful to inhibit an immune responsecomprising a pharmaceutically effective amount of a soluble ligand whichrecognizes and binds a gp39 antigen and an acceptable carrier.
 44. Apharmaceutical composition useful to inhibit an immune responsecomprising a pharmaceutically effective amount of a combination of asoluble ligand which recognizes and binds a B7 antigen and a solubleligand which recognizes and binds a gp39 antigen and an acceptablecarrier.
 45. A pharmaceutical composition useful to inhibit an immuneresponse comprising a pharmaceutically effective amount of a combinationof a soluble ligand which recognizes and binds a CD28 antigen and asoluble ligand which recognizes and binds a CD40 antigen and anacceptable carrier.
 46. A pharmaceutical composition useful to inhibitan immune response comprising a pharmaceutically effective amount of acombination of a soluble ligand which recognizes and binds a CTLA4antigen and a soluble ligand which recognizes and binds a CD40 antigenand an acceptable carrier.
 47. A pharmaceutical composition useful toinhibit an immune response comprising a pharmaceutically effectiveamount of a combination of a soluble ligand which recognizes and binds agp39 antigen and a soluble ligand which recognizes and binds a CD28antigen and an acceptable carrier.
 48. A pharmaceutical compositionuseful to inhibit an immune response comprising a pharmaceuticallyeffective amount of a combination of a soluble ligand which recognizesand binds a gp39 antigen and a soluble ligand which recognizes and bindsa CTLA4 antigen and an acceptable carrier.
 49. A pharmaceuticalcomposition useful to inhibit an immune response comprising apharmaceutically effective amount of a combination of a soluble ligandwhich recognizes and binds a B7 antigen and a soluble ligand whichrecognizes and binds a CD40 antigen and an acceptable carrier.
 50. Amethod of inhibiting an immune response in a subject comprisingadministering to the subject an amount of the pharmaceutical compositionof claim 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, or 49 effective toinhibit the immune response in the subject.